CN101392230A - Recombinant escherichia coli for expression of adenomethionine synthetase - Google Patents
Recombinant escherichia coli for expression of adenomethionine synthetase Download PDFInfo
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- CN101392230A CN101392230A CNA2008100557043A CN200810055704A CN101392230A CN 101392230 A CN101392230 A CN 101392230A CN A2008100557043 A CNA2008100557043 A CN A2008100557043A CN 200810055704 A CN200810055704 A CN 200810055704A CN 101392230 A CN101392230 A CN 101392230A
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Abstract
The invention provides a recomposed large intestine Escherichia coli expressing adenosine methionine synthetase with CGMCC No. 2299 and the composition method of the recomposed large intestine Escherichia coli. The invention further provides a recomposed expression carrier pMETK of adenosine methionine synthetase. In the recomposed large intestine Escherichia coli expressing adenosine methionine synthetase with CGMCC No. 2299, the expression quantity of the adenosine methionine synthetase accounts for 31.36 percent of soluble albumen of the strain, the activity of the adenosine methionine synthetase expressed is as high as 7.38U/ml, far higher than that of wild type strains.
Description
Technical field
The present invention relates to biological technical field, relate to the recombinant escherichia coli and the application thereof of an expression of adenomethionine synthetase particularly.
Background technology
China is populous nation, also is " hepatitis big country " simultaneously.According to national viral hepatitis epidemiology survey in 1992, the whole nation had 1.2 hundred million people to carry hepatitis B virus approximately, and hepatitis C infection rate in general crowd is 3.1%.China has 2,000 ten thousand routine chronic hepatitis B patients now, and wherein part might be converted into liver cirrhosis, even liver cancer.
Ademetionine (SAM) is a kind of very important mesostate in the human body.It can generate anti-oxidant and detoxifcation material---halfcystine crucial in the body by changeing the sulphur approach, and another kind of crucial anti-oxidant and toxicide matter---the gsh of regeneration is removed the oxide compound stress situation in the liver, thereby reached the purpose of preventing and treating hepatopathy.SAM can also promote the synthetic of dependency fat phosphatide in addition, and the ratio of reducing cholesterol and phosphatide is recovered flowability of cell membranes, promotes the secretion of biliary active transport and R-Glyceric acid three fat.Therefore SAM has better curative effect to hepatic diseases such as intrahepatic cholestasis, various acute and chronic hepatitis and fatty liver, liver cirrhosis.SAM appeared on the European medical market the eighties in 20th century, and U.S. FDA official approval SAM listing in 1999 makes it become one of dietary supplements salable rapidly, and annual sales amount is above 1,000,000,000 dollars.Mechanism is clear, effect is remarkable, rapid-action, side effect is little because it is treated, and demand at home is also increasing in recent years, and vast market prospect is arranged.
At present, the production method of SAM mainly contains 3 kinds of chemical synthesis, fermentation method and Enzymatic transformation methods.
Chemical synthesis adopts S one adenosyl homocysteine (S-adenosythomocysteine) and methyl donor (CH3I) to synthesize, ademetionine has (+) and (one) two kind of configuration, have only (one) configuration that biological activity is just arranged, but contain a large amount of (+) gland former times methionine(Met) in the synthetic product, and be difficult to separate.Fermentation method adopts and add the L-methionine(Met) in substratum, with yeast fermentation production (one) type ademetionine, is the main method of producing the general methionine(Met) of gland the sixties to the eighties.The Enzymatic transformation method mainly utilizes adenomethionine synthase catalytic substrate L one methionine(Met) and ATP to generate (one) type gland former times methionine(Met).Enzyme has quick, single-minded characteristics as biological catalyst, so the Enzymatic transformation method is produced, and ademetionine has efficiently, technology is simple, the product biological activity is high and free of contamination advantage.
Enzymic synthesis is applied to suitability for industrialized production, obtaining the adenomethionine synthase that catalysis uses in a large number becomes key, and the difficult problem of large-scale production adenomethionine synthase is perplexing the industrial applications of Enzymatic transformation method always.The present invention adopts the genetically engineered recombinant technology, has obtained to efficiently express the engineering bacteria of adenomethionine synthase, and is significant to the suitability for industrialized production of enzyme-catalyzed change ademetionine.
Summary of the invention
(1) technical problem that will solve
The purpose of this invention is to provide the recombinant escherichia coli and the application thereof of an expression of adenomethionine synthetase, another purpose of the present invention provides a kind of recombinant expression vector and application thereof of adenomethionine synthase.
(2) technical scheme
The invention provides recombinant escherichia coli (Escherichia coli) the CGMCC No.2299 of an expression of adenomethionine synthetase.This bacterial strain has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on December 26th, 2007, be called for short CGMCC, and deposit number is CGMCC No.2299.
The construction process of this recombinant escherichia coli comprises the steps:
(1) the metK gene of usefulness PCR method amplification coding colon bacillus adenomethionine synthase;
(2) with the PCR product cloning that obtains to connection carrier pBR322, obtain recombinant expression vector pMETK;
(3) recombinant expression vector pMETK is transformed colon bacillus DH5 α, screen positive transformant, obtain expressing the recombinant escherichia coli of adenomethionine synthase.
The present invention also provides a kind of recombinant expression vector pMETK of adenomethionine synthase, and it is that metK gene clone with coding colon bacillus adenomethionine synthase obtains to connection carrier pBR322.
Recombinant escherichia coli of the present invention (Escherichia coli) CGMCC No.2299 can efficiently express adenomethionine synthase, can be applicable to the industrialization synthesizing adenosine methionine.
Recombinant expression vector pMETK of the present invention also can be applicable to the industrialization synthesizing adenosine methionine.
(3) beneficial effect
The recombinant escherichia coli of expression adenomethionine synthase provided by the invention (Escherichiacoli) CGMCC No.2299, the expression amount of its adenomethionine synthase accounts for 31.36% of thalline soluble proteins, and is higher 16 times than wild bacterium; The adenomethionine synthase activity of expressing is up to 7.38U/ml, and is higher 22 times than wild bacterium.
Bacterial strain of the present invention has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on December 26th, 2007, be called for short CGMCC, and deposit number is CGMCC No.2299.
Description of drawings
Fig. 1 is the structure iron of recombinant expression vector pMETK;
Fig. 2 is the electrophorogram of Pst I and EcoR I double digestion recombinant expression vector pMETK, wherein 1 expression sample;
Fig. 3 SDS-PAGE detects the electrophorogram of expressing quantity, wherein, and 1 and 3 expression samples, 2 expression SDS-PAGE low molecular weight protein (LMWP) Marker.
Embodiment
Following examples are used to illustrate the present invention, but are not used for limiting the scope of the invention.
Used main agents and equipment in the experiment
1, main raw
DNA extraction agent box is purchased in QIAGEN company (DNeasy Tissue Kit, catalog number 69504); Cloning vector pBR322, colon bacillus DH5 α purchase the company in Xenogen; Restriction enzyme Pst I, EcoR I, the Taq archaeal dna polymerase, the T4 dna ligase, N,O-Diacetylmuramidase and relevant damping fluid are purchased the company in Promega; The used peptone of LB substratum, yeast powder are purchased the company in OXOID; All the other reagent are commercial, are analytical pure.
2, major equipment
Desk centrifuge: Sigma company; Constant temperature shaking table: Infors company; Electrophoresis apparatus: Bio-Rad company; Ultraviolet projectoscope and imaging device thereof: Pharmacia Corp; Pcr amplification instrument: u.s.a. applied biosystem company.
One, the structure of recombinant expression vector pMETK:
The metK gene discloses (J.Biol.Chem.259 (23), 14505-14507 (1984)) in the literature
(1) E.coli DH5 α is inoculated on the LB solid medium, cultivated 18 hours for 37 ℃, the toothpick picking list colony inoculation of using sterilization is in 50ml LB liquid nutrient medium then, and 37 ℃, 180rpm shaking table overnight incubation are extracted its genomic dna with DNA extraction agent box then.
(2) entrust Shanghai to give birth to the synthetic following primer of worker's biotechnology company limited:
The forward primer sequence:
5’-AA
CTGCAGAGTCGTGGTAGGATCCGCTACCACA-3’，
The reverse primer sequence:
5’-G
GAATTCATCTGCAATAAACGGCAGTGCGC-3’；
(3) get DNA masterplate 1 μ g, add each the 2 μ mol/L of primer in the step (2) respectively, Taq archaeal dna polymerase 5IU, dNTPs 200 μ mol/L, 1 * PCR damping fluid, 20 μ L, mix, carry out pcr amplification by the following method: 94 ℃ of sex change 1min, (94 ℃ of 30s, 55 ℃ of 30s circulate 30 times, 72 ℃ of 2.5min), extend 10min then.Ordinary method by " molecular cloning " reclaims goal gene metK.
(4) get the solution that contains 1 μ g above-mentioned purpose gene, add 10 * Pst I enzyme cutting buffering liquid, 2 μ l successively, EcoR I enzyme cutting buffering liquid 2 μ l, be settled to 18 μ l with sterilized water, add Pst I restriction enzyme (5U/ μ l) 1 μ l more respectively, EcoR I restriction enzyme (5U/ μ l) 1 μ l mixes, and places 37 ℃ of water-bath insulations 3 hours;
(5) the metK gene fragment in the reaction solution of electrophoresis recycling step (4);
(6) get 1 μ g plasmid pBR322, add 10 * PstI enzyme cutting buffering liquid, 2 μ l successively, EcoR I enzyme cutting buffering liquid 2 μ l, be settled to 18 μ l with sterilized water, add Pst I restriction enzyme (5U/ μ l) 1 μ l more respectively, EcoR I restriction enzyme (5U/ μ l) 1 μ l mixes, and places 37 ℃ of water-bath insulations 3 hours;
(7) electrophoresis reclaims the pBR322 plasmid after enzyme is cut;
(8) get 0.1 μ g through the plasmid pBR322 of above-mentioned double digestion and the metK gene uniform mixing of 0.12 μ g step (4) recovery, add sterilized water 8 μ L, 45 ℃ of insulation 5min, add 10 * T4DNA ligase enzyme damping fluid, 1 μ L, T4 dna ligase (5U/ μ l) 0.5 μ L then, mix the back in 16 ℃ of insulations 24 hours, obtain recombinant expression vector pMETK, its structure iron as shown in Figure 1;
Two, the structure of recombinant escherichia coli
(1) the single bacterium colony of picking E.coli DH5 α from the LB solid medium flat board, be inoculated in the 5ml LB liquid nutrient medium, 37 ℃ of shaking tables were cultivated 12 hours, were inoculated in the 100ml LB liquid nutrient medium with 1:50 then, 37 ℃ of shaking tables were cultivated 3 hours, to OD600=0.5;
(2) place ice bath to cool off 10min nutrient solution, the centrifugal collection thalline of 3000rpm, the CaCl of the 0.05mol/L of usefulness precooling
2The solution 10ml thalline that suspends gently places ice bath to cool off 30min again, and 3000rpm collects thalline, contains 15% 0.05mol/LCaCl with 4ml then
2The solution suspension cell places ice bath stand-by;
(3) get the competent cell suspension uniform mixing that above-mentioned recombinant expression vector pMETK2 μ L and 200 μ L steps (2) obtain, ice bath 30min transfers to then in 37 ℃ of water-baths and heats 5min, places ice bath to cool off 5min again;
(4) get 100 μ L conversion reaction stostes, be coated on the LB solid medium that contains 200 μ g/ml oxidation tsiklomitsins, cultivate half an hour, absorb fully for 37 ℃ until liquid with aseptic glass stick;
(5) be inverted flat board, 37 ℃ of constant temperature culture 16 hours, take out dull and stereotyped during the single bacterium colony waiting to occur obviously and do not overlap each other, single bacterium colony on the picking flat board, screening adenomethionine synthase expression amount the highest positive transformant (measuring method of expression of enzymes amount is seen embodiment 2), sieve at last recombinant escherichia coli (Escherichia coli) CGMCCNo.2299 of expression adenomethionine synthase of the present invention.
Wherein, the authentication method of positive transformant is as follows:
1. double digestion is identified:
A. get the transformant of picking in the step (5), use the LB liquid culture, the bacterium liquid 1.5ml that obtains, centrifugal 30 seconds of 12000rpm, supernatant discarded is with 120ml STET damping fluid (0.1mol/L NaCl; 10mmol/L TrisCl, PH8.0; 10mmol/L EDTA, PH8.0; 5% Triton X-100) resuspended thalline;
B. the lysozyme soln 10ml that adds 10mg/ml in the thalline suspension vibrates to be placed in 3 seconds in the boiling water and heated 50 seconds, and 12000rpm is centrifugal, supernatant discarded and bacterial chip;
C. in above-mentioned precipitation, add 10 * Pst I enzyme cutting buffering liquid, 2 μ l successively, EcoR I enzyme cutting buffering liquid 2 μ l, be settled to 18 μ l with sterilized water, add Pst I restriction enzyme (5U/ μ l) 1 μ l more respectively, EcoR I restriction enzyme (5U/ μ l) 1 μ l, mix, place 37 ℃ of water-bath insulations 3 hours;
D. above endonuclease reaction liquid is carried out electrophoresis detection, obtain two bands (seeing accompanying drawing 2) that size is about 1.8kb and 3.6kb, in full accord with expected results.
2. gene sequencing is identified:
By the recombinant expression plasmid pMETK in the transformant in " molecular cloning " described classical way extraction, complete sequence analysis is carried out at center, the genoid group of the making a gift to someone north, records length overall 5407bp, and is in full accord with expected results.Concrete outcome is as follows:
1 TCATGTTTGACAGCTTATCATCGATAAGCTTTAATGCGGTAGTTTATCACAGTTAAATTG
61 CTAACGCAGTCAGGCACCGTGTATGAAATCTAACAATGCGCTCATCGTCATCCTCGGCAC
121 CGTCACCCTGGATGCTGTAGGCATAGGCTTGGTTATGCCGGTACTGCCGGGCCTCTTGCG
181 GGATATCGTCCATTCCGACAGCATCGCCAGTCACTATGGCGTGCTGCTAGCGCTATATGC
241 GTTGATGCAATTTCTATGCGCACCCGTTCTCGGAGCACTGTCCGACCGCTTTGGCCGCCG
301 CCCAGTCCTGCTCGCTTCGCTACTTGGAGCCACTATCGACTACGCGATCATGGCGACCAC
361 ACCCGTCCTGTGGATCCTCTACGCCGGACGCATCGTGGCCGGCATCACCGGCGCCACAGG
421 TGCGGTTGCTGGCGCCTATATCGCCGACATCACCGATGGGGAAGATCGGGCTCGCCACTT
481 CGGGCTCATGAGCGCTTGTTTCGGCGTGGGTATGGTGGCAGGCCCCGAGGCCGGGGGACT
541 GTTGGGCGCCATCTCCTTGCATGCACCATTCCTTGCGGCGGCGGTGCTCAACGGCCTCAA
601 CCTACTACTGGGCTGCTTCCTAATGCAGGAGTCGCATAAGGGAGAGCGTCGACCGATGCC
661 CTTGAGAGCCTTCAACCCAGTCAGCTCCTTCCGGTGGGCGCGGGGCATGACTATCGTCGC
721 CGCACTTATGACTGTCTTCTTTATCATGCAACTCGTAGGACAGGTGCCGGCAGCGCTCTG
781 GGTCATTTTCGGCGAGGACCGCTTTCGCTGGAGCGCGACGATGATCGGCCTGTCGCTTGC
841 GGTATTCGGAATCTTGCACGCCCTCGCTCAAGCCTTCGTCACTGGTCCCGCCACCAAACG
901 TTTCGGCGAGAAGCAGGCCATTATCGCCGGCATGGCGGCCGACGCGCTGGGCTACGTCTT
961 GCTGGCGTTCGCGACGCGAGGCTGGATGGCCTTCCCCATTATGATTCTTCTCGCTTCCGG
1021 CGGCATCGGGATGCCCGCGTTGCAGGCCATGCTGTCCAGGCAGGTAGATGACGACCATCA
1081 GGGACAGCTTCAAGGATCGCTCGCGGCTCTTACCAGCCTAACTTCGATCATTGGACCGCT
1141 GATCGTCACGGCGATTTATGCCGCCTCGGCGAGCACATGGAACGGGTTGGCATGGATTGT
1201 AGGCGCCGCCCTATACCTTGTCTGCCTCCCCGCGTTGCGTCGCGGTGCATGGAGCCGGGC
1261 CACCTCGACCTGAATGGAAGCCGGCGGCACCTCGCTAACGGATTCACCACTCCAAGAATT
1321 GGAGCCAATCAATTCTTGCGGAGAACTGTGAATGCGCAAACCAACCCTTGGCAGAACATA
1381 TCCATCGCGTCCGCCATCTCCAGCAGCCGCACGCGGCGCATCTCGGGCAGCGTTGGGTCC
1441 TGGCCACGGGTGCGCATGATCGTGCTCCTGTCGTTGAGGACCCGGCTAGGCTGGCGGGGT
1501 TGCCTTACTGGTTAGCAGAATGAATCACCGATTCGCGAGCGAACGTGAAGCGACTGCTGC
1561 TGCAAAACGTCTGCGACCTGAGCAACAACATGAATGGTCTTCGGTTTCCGTGTTTCGTAA
1621 AGTCTGGAAACGCGGAAGTCAGCGCCCTGCACCATTATGTTCCGGATCTGCATCGCAGGA
1681 TGCTGCTGGCTACCCTGTGGAACACCTACATCTGTATTAACGAAGCGCTGGCATTGACCC
1741 TGAGTGATTTTTCTCTGGTCCCGCCGCATCCATACCGCCAGTTGTTTACCCTCACAACGT
1801 TCCAGTAACCGGGCATGTTCATCATCAGTAACCCGTATCGTGAGCATCCTCTCTCGTTTC
1861 ATCGGTATCATTACCCCCATGAACAGAAATCCCCCTTACACGGAGGCATCAGTGACCAAA
1921 CAGGAAAAAACCGCCCTTAACATGGCCCGCTTTATCAGAAGCCAGACATTAACGCTTCTG
1981 GAGAAACTCAACGAGCTGGACGCGGATGAACAGGCAGACATCTGTGAATCGCTTCACGAC
2041 CACGCTGATGAGCTTTACCGCAGCTGCCTCGCGCGTTTCGGTGATGACGGTGAAAACCTC
2101 TGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGA
2161 CAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCGCAGCCATGACCCAG
2221 TCACGTAGCGATAGCGGAGTGTATACTGGCTTAACTATGCGGCATCAGAGCAGATTGTAC
2281 TGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCA
2341 TCAGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGC
2401 GAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACG
2461 CAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGT
2521 TGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAA
2581 GTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCT
2641 CCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCC
2701 CTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGG
2761 TCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCT
2821 TATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAG
2881 CAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGA
2941 AGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGA
3001 AGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTG
3061 GTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAG
3121 AAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAG
3181 GGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAAT
3241 GAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCT
3301 TAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGAC
3361 TCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAA
3421 TGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCG
3481 GAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATT
3901 CACCGTTCGCGAAATTGGCTATGTGCATTCCGACATGGGCTTTGACGCTAACTCCTGTGC
3961 GGTTCTGAGCGCTATCGGCAAACAGTCTCCTGACATCAACCAGGGCGTTGACCGTGCCGA
4021 TCCGCTGGAACAGGGCGCGGGTGACCAGGGTCTGATGTTTGGCTACGCAACTAATGAAAC
4081 CGACGTGCTGATGCCAGCACCTATCACCTATGCACACCGTCTGGTACAGCGTCAGGCTGA
4141 AGTGCGTAAAAACGGCACTCTGCCGTGGCTGCGCCCGGACGCGAAAAGCCAGGTGACTTT
4201 TCAGTATGACGACGGCAAAATCGTTGGTATCGATGCTGTCGTGCTTTCCACTCAGCACTC
4261 TGAAGAGATCGACCAGAAATCGCTGCAAGAAGCGGTAATGGAAGAGATCATCAAGCCAAT
4321 TCTGCCCGCTGAATGGCTGACTTCTGCCACCAAATTCTTCATCAACCCGACCGGTCGTTT
4381 CGTTATCGGTGGCCCAATGGGTGACTGCGGTCTGACTGGTCGTAAAATTATCGTTGATAC
4441 CTACGGCGGCATGGCGCGTCACGGTGGCGGTGCATTCTCTGGTAAAGATCCATCAAAAGT
4501 GGACCGTTCCGCAGCCTACGCAGCACGTTATGTCGCGAAAAACATCGTTGCTGCTGGCCT
4561 GGCCGATCGTTGTGAAATTCAGGTTTCCTACGCAATCGGCGTGGCTGAACCGACCTCCAT
4621 CATGGTAGAAACTTTCGGTACTGAGAAAGTGCCTTCTGAACAACTGACCCTGCTGGTACG
4681 TGAGTTCTTCGACCTGCGCCCATACGGTCTGATTCAGATGCTGGATCTGCTGCACCCGAT
4741 CTACAAAGAAACCGCAGCATACGGTCACTTTGGTCGTGAACATTTCCCGTGGGAAAAAAC
4801 CGACAAAGCGCAGCTGCTGCGCGATGCTGCCGGTCTGAAGTAATCTTTCTTCACCTGCGT
4861 TCAAAGGCCAGCCTCGCGCTGGCCTTTTTCTTTTGGATAGGCGTTCACGCCGCATCCGGC
4921 AAAAAAACCGCCCGCACAATAACATCATTCTTCCTGATCACGTTTCACCGCAGATTATCA
4981 TCACAACTGAAACCGATTACACCAACCACAACAGACAAAGATTTGTAATATTTTCATATT
5041 ATTATTCGGTTTTCACAGTTGTTACATTTCTTTTCAGTAAAGTCTTAATTGCAGATAACA
5101 GCGTTTAATCTATGATGATATAACTCAATTATTTTCATGCACTTAAATCATAACTAAGAT
5161 AAATGTTAGTGTAAGCGATTACACTGATGTGATTTGCTTCACATCTTTTTACGTCGTACT
5221 CACCTATCTTAATTCACAATAAAAAATAACCATATTGGAGGGCATCATGCCTGACGCTAA
5281 AAAACAGGGGCGGTCAAACAAGGCAATGACGTTTTTCGTCTGCTTCCTTGCCGCTCTGGC
5341 GGGATTACTCTTTGGCCTGGATATCGGTGTAATTGCTGGCGCACTGCCGTTTATTGCAGA
5401 TGAATT
The expression amount and the determination of activity of the recombinant escherichia coli adenomethionine synthase that embodiment 2 makes up
Experiment
(1) mensuration of adenomethionine synthase expression amount
Single bacterium colony of the recombinant escherichia coli that picking embodiment 1 obtains (Escherichia coli) CGMCCNo.2299, be inoculated into 10ml liquid LB substratum (the oxidation tsiklomitsin that contains 25 μ g/ml), shaking table was cultivated 6 hours, sampling is measured the wherein expression amount of adenomethionine synthase with the SDS-PAGE polyacrylamide gel electrophoresis, electrophoresis result as shown in Figure 3, the expression amount of SAM synthetic enzyme accounts for 31.36% of thalline soluble proteins.
(2) determination of activity of adenomethionine synthase
Preparation reaction solution: by 75mmol Tris-HCl (PH8.0), 250mmol KCl, 9mmol MgCl
2, 60 μ mol methionine(Met), 5mmol ATP and 1ml crude enzyme liquid form the reaction system of 100ml, behind 37 ℃ of reaction 30min, add perchloric acid to final concentration and be 0.3% and make the stopping of reaction, with the amount of ademetionine in the HPLC external standard method reaction solution.The unit of activity of enzyme is defined as: under above reaction conditions, it is a unit of activity that 30min catalysis forms the needed enzyme amount of 1 μ mol ademetionine.
Through measuring, the activity of the adenomethionine synthase that recombinant escherichia coli (Escherichia coli) CGMCC No.2299 expresses is 7.38U/ml.
Sequence table
＜110〉Beijing KaiYin Bioisystech Co., Ltd
＜120〉recombinant escherichia coli of an expression of adenomethionine synthetase
<130>?K0801
<160>?1
<170>?PatentIn?version?3.2
<210>?1
<211>?5046
<212>?DNA
＜213〉colon bacillus (Escherichia coli)
<400>?1
Claims (5)
1, the recombinant escherichia coli of an expression of adenomethionine synthetase (Escherichia coli) CGMCC No.2299.
2, a kind of recombinant expression vector pMETK of adenomethionine synthase is characterized in that it is that metK gene clone with coding colon bacillus adenomethionine synthase obtains to connection carrier pBR322.
3, the construction process of recombinant escherichia coli according to claim 1 is characterized in that it comprises the steps:
(1) the metK gene of usefulness PCR method amplification coding colon bacillus adenomethionine synthase;
(2) with the PCR product cloning that obtains to connection carrier pBR322, obtain the described recombinant expression vector pMETK of claim 2;
(3) recombinant expression vector pMETK is transformed colon bacillus DH5 α, screen positive transformant, obtain expressing the recombinant escherichia coli of adenomethionine synthase.
4, the application of recombinant escherichia coli according to claim 1 in synthesizing adenosine methionine.
5, the application of recombinant expression vector according to claim 2 in synthesizing adenosine methionine.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101870964A (en) * | 2010-05-25 | 2010-10-27 | 北京凯因科技股份有限公司 | Method for improving SAM synthetase expression level |
| CN116042561A (en) * | 2022-11-11 | 2023-05-02 | 山东理工大学 | S-adenosylmethionine synthetase mutant and application thereof |
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| CN1191369C (en) * | 2002-06-14 | 2005-03-02 | 中国科学院上海生命科学研究院生物化学与细胞生物学研究所 | Process for producing adenosylmethionine by metabolic engineering bacteria |
| DE10309856A1 (en) * | 2003-03-06 | 2004-09-23 | Consortium für elektrochemische Industrie GmbH | Process for the fermentative production of S-adenosylmethionine |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101870964A (en) * | 2010-05-25 | 2010-10-27 | 北京凯因科技股份有限公司 | Method for improving SAM synthetase expression level |
| CN101870964B (en) * | 2010-05-25 | 2012-07-04 | 北京凯因科技股份有限公司 | Method for improving SAM synthetase expression level |
| CN116042561A (en) * | 2022-11-11 | 2023-05-02 | 山东理工大学 | S-adenosylmethionine synthetase mutant and application thereof |
| CN116042561B (en) * | 2022-11-11 | 2023-10-24 | 山东理工大学 | S-adenosylmethionine synthetase mutant and application thereof |
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| CN101392230B (en) | 2012-05-23 |
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